Steroid hormones such as cortisol are key regulators of a diverse array of biological processes that evoke physiological changes by binding to members of the nuclear receptor super family of transcription factors. Because glucocorticoids control the expression of numerous genes, multiple mechanisms are used to ensure tight control over hormone production. Our preliminary findings provide evidence for the role of adrenocorticotropin (ACTH)-stimulated, microtubule-dependent mitochondrial trafficking in maintaining optimal glucocorticoid output. We have also identified several proteins, including the GTPase RhoA and the Rho effector diaphanous 1 (DIAPH1), that act to facilitate this movement. ACTH signaling controls the ability of DIAPH1 to interact with RhoA and several other binding partners. However, the functional significance of these interactions has not fully been elucidated, nor have we defined if ACTH-stimulated mitochondrial movement enables cortisol production by facilitating the delivery of substrate between the ER and mitochondria. We hypothesize that ACTH-stimulated increases in the rate of mitochondrial trafficking are required for inter- organelle substrate exchange and glucocorticoid biosynthesis. To test this hypothesis we will define the mechanism by which ACTH signaling controls DIAPH1 complex assembly (Specific Aim 1). We have generated a stable cell line that expresses shRNA against DIAPH1 to define the role of the protein in mitochondrial movement and hormone production. We will also assess the functional significance of the interactions between DIAPH1 and several binding partners, including tubulin, kinesin, and the oxysterol- binding-protein related protein 2 (ORP2). Studies will also be performed to define the role of phosphorylation in controlling DIAPH1 function (Specific Aim 2). We have found that ACTH signaling stimulates the phosphorylaton of DIAPH1. The role of this post-translational modification on DIAPH1 function will be examined using phospho-mutants and a phospho-specific antibody. Finally, we will determine the mechanism by which 11-deoxycortisol is transported between the ER and mitochondria (Specific Aim 3). We have found that ORP2 co-purifies with DIAPH1 and hypothesize that this lipid binding protein may facilitate substrate transfer between the ER and mitochondria. We will characterize the role of ORP2 in cortisol biosynthesis and also determine if fusion between ER and mitochondria play a role in inter-organelle substrate delivery. The knowledge gained from these studies will provide valuable information about a largely unexplored facet of steroid hormone synthesis in the adrenal cortex. Our findings may also provide insight into the mechanism by which substrates are transferred between the ER and mitochondria in other metabolic processes. PUBLIC HEALTH RELEVANCE: Understanding how genes are regulated in cells that make steroid hormones will provide insight into the mechanisms by which pathophysiological concentrations of cortisol and adrenal androgens are produced. This work will provide insight into multiple endocrine disorders, including adrenal hyperplasia, polycystic ovary syndrome, and Cushing's disease.